GENERAL OVERVIEW: The Flow Cytometry Core at NEI provides flow cytometry analytical and sorting equipment and services to the NEI Intramural community. It utilizes and develops state-of-the-art sample preparation, data acquisition and analysis, and sorting procedures in collaborative research projects. Provides training to students, fellows, and principal investigators on sample preparation, staining, and post-sort handling. Assesses technical research needs and recommends recruitment of the appropriate staff and acquisition of the equipment needed to meet those needs. The 2 sorters, FACS Aria II and FACS Aria Fusion, are largely operated by trained Core personnel. The analytical instruments, 2 MACS Quant instruments and the BD FACS Calibur, are largely operated by users who receive training from Core personnel. SERVICES PROVIDED THE CORE: This year, sixty-one individuals from thirteen different laboratories used the facility. These services and collaborative services were performed for 8 Principal Investigators (PIs) from 8 NEI labs (ERPD, LI, LRCMB, MSF, N-NRL, RN, OGVFB and UNGIRD), plus 3 PIs from 3 other institutes at NIH (NICHD, NIDCD and NIDDK). Over 34,429 samples were analyzed. This year the core performed about 1,423 hours of sorting. Among the techniques now in use within the core are methods for phenotyping live cells, detecting gene expression, monitoring membrane and DNA content changes due to apoptosis or proliferation, measurement of intracellular proteins and quantification of soluble proteins. The work involving human tissues includes the sorting of peripheral blood mononuclear cells to study their cytokine production, genotype and DNA or RNA expression. The sources are blood, buffy coat, and white cells. Some analytical work had been done with eye fluids, eye tissue specimen, protein, and tears. The National Eye Institute made a great investment in biosafety with the addition BD FACSAria Fusion flow cytometer equipped with a fully integrated biosafety cabinet. This sorter meets the recent NIH's operator and sample protection requirements as well as global standards for bioprotection for processing human samples. No human tissues were stored by the core. The Core encourages, but does not obligate, users to acknowledge the Core contribution in their publications. The following are examples of the publications that acknowledged the use of NEI Flow Cytometry Core resources: Wandu WS, Tan C, Ogbeifun O, Vistica BP, Shi G, Hinshaw SJH, et al. (2015) Leucine-Rich Repeat Kinase 2 (Lrrk2) Deficiency Diminishes the Development of Experimental Autoimmune Uveitis (EAU) and the Adaptive Immune Response. PLoS ONE 10(6): e0128906. doi:10.1371/journal.pone.0128906 PMID: 26067490 Silver PB, Horai R, Chen J, Jittayasothorn Y, Chan CC, Villasmil R, Kesen MR, Caspi RR. Retina-specific T regulatory cells bring about resolution and maintain remission of autoimmune uveitis. J Immunol. 2015 Apr 1;194(7):3011-9. doi: 10.4049/jimmunol.1402650. Epub 2015 Feb 25. PMID: 25716996 Chen P, Tucker W, Hannes S, Liu B, Si H1, Gupta A, Lee RW, Sen HN, Nussenblatt RB. Levels of blood CD1c+ mDC1 and CD1chi mDC1 subpopulation reflect disease activity in noninfectious uveitis. Invest Ophthalmol Vis Sci. 2014 Dec 16;56(1):346-52. doi: 10.1167/iovs.14-15416. PMID: 25515573 Mattapallil MJ, Silver PB, Cortes LM, St Leger AJ, Jittayasothorn Y, Kielczewski JL, Moon JJ, Chan CC1, Caspi RR. Characterization of a New Epitope of IRBP That Induces Moderate to Severe Uveoretinitis in Mice With H-2b Haplotype. Invest Ophthalmol Vis Sci. 2015 Aug 1;56(9):5439-49. doi: 10.1167/iovs.15-17280. PMID: 26284549 Horai R, Zrate-Blads CR, Dillenburg-Pilla P, Chen J, Kielczewski JL, Silver PB, Jittayasothorn Y, Chan CC, Yamane H, Honda K, Caspi RR. Microbiota-Dependent Activation of an Autoreactive T Cell Receptor Provokes Autoimmunity in an Immunologically Privileged Sitee. Immunity. 2015 Aug 18;43(2):343-53. doi: 10.1016/j.immuni.2015.07.014. PMID: 26287682 Chong WP, van Panhuys N, Chen J, Silver PB, Jittayasothorn Y, Mattapallil MJ, Germain RN, Caspi RR (2015) NK-DC crosstalk controls the autopathogenic Th17 response through an innate IFNg-IL-27 axis. J Exp Med, in press (e-pub ahead of print). TRAINING: More than twenty users from different labs received training how to operate the MACSQuant instrumentation. The availability of the Technical IRTA position continues to have a very positive effect on the Core operation. There is a great need for well-trained flow cytometry operators. Through the TechIRTA, the hours of operation of the facility have been expanded from 40 hr/week to 60 hr/week. The expanded hours of operation helped to reduce the backlog on the sorter schedule. The TechIRTA position has greatly enhanced the ability of the Core to use the cell sorter for high throughput multicolor analysis. This year the core hosted a summer student from the Diversity In Vision Research and Ophthalmology (DIVRO) summer internship program. This student had special needs due to profound deafness. The Guidelines For Mentoring an NIH Trainee Who Is Deaf or Hard of Hearing, published by Office of the Director for Intramural Research, were used to help mentor this student. The student worked on Extracting High-Dimensional Single Cell Data through Flow Cytometry Analysis of Eye Immune Cells with Novel Bioinformatics Tools and presented a poster at the NEI Summer Interns Poster Day. Several formal training sessions were offered by the core to the NEI community. These courses included: Introduction to Flow Cytometry, Advanced Techniques (Ex. Apoptosis applications), Analytical Instrument Operation and Data Analysis with FloJo Software. The Core Manager completed 32 hours of continuing education in management of core laboratories. The technical IRTA completed 24 hours of formal training outside the Institute.